1. Field of the Invention
This invention relates generally to a method and a kit for separating double-stranded nucleic acid molecules from a mixture containing both single-stranded and double-stranded nucleic acid molecules and is particularly suitable for separating hybridized from unhybridized probe nucleic acids.
2. Description of the Prior Art
An important tool in the rapidly-evolving fields of genetic engineering, environmental microbial contamination monitoring, and medical diagnostics is the use of DNA and RNA probes. DNA and RNA probes are single-stranded nucleic acid molecules generally synthesized by so-called gene machines or made using recombinant DNA methods. Probes are constructed so that the base (i.e., gene) sequences of the probe match (and lend themselves to hybridization with) complementary sequences on a target molecule. They are used for clinical diagnosis of genetic disorders, cancer, and disease-causing bacteria, protozoans, and viruses. In the environmental field, they can be used to rapidly identify specific microbial contaminants. Research scientists use probes in recombinant DNA experiments and in gene activation studies.
Prior to use, each molecule of the probe is labelled with a marker, such as a radioactive tag, in order to allow determination of when and where hybridization has occurred.
Before the hybridized target nucleic acid molecules can be identified based upon the presence of the labelled probe, the unhybridized single-stranded labelled probe molecules must be separated out. Otherwise, this latter material will act as "background noise" inhibiting attempts to identify the hybridized material. Conventionally utilized commercial methods for effecting this separation of hybridized probe molecules from unhybridized single-stranded probe molecules usually involve iramobilizing the target nucleic acid on a membrane which is reactive with nucleic acids. Such membranes bind all nucleic acids nonselectively. In view of this nonselectivity characteristic, the active sites on the membrane must be blocked before probe nucleic acid is added. This blocking entails numerous steps and an incubation period typically of two hours. Thus, the membrane must be treated with various blocking agents after the target DNA is affixed to prevent nonspecific adhesion of the probe nucleic acid so that washing the membrane after hybridization will remove unhybridized probe. Alternatively, selective absorption of double-stranded nucleic acids using hydroxylapatite is employed. The hydroxylapatite serves to effectively separate double-from single-stranded DNA. This capability results from hydroxylapatite's selective affinity for place in a single phase. Commercial kits using conditions.
Either radioactive or nonradioactive probes may be used with the membrane immobilization technique. In a nutshell, the radioactive probe method first involves incubating a membrane, that has single-stranded target sequences attached, with a radioactively labelled probe (usually phosphorus-32 (.sup.32 P)) consisting of a single strand of DNA or RNA with base sequences that are possibly complementary to the target sequences being studied. The probe hybridizes with only those target nucleic acids containing a complementary nucleic acid sequence. After hybridization, the membrane is washed and hybrids are detected by autoradiography. The presence of characteristic hybrid nucleic acid on the autoradiogram is indicative of the presence of a specific target sequence. Typically, the assay of a hybridized radioactively labelled probe requires numerous steps and 40 hours. The extensive time and effort result from the necessity of binding target nucleic acid to the membrane, blocking the remaining sites on the membrane that would otherwise nonspecifically bind labelled probe, hybridizing for long periods of time because the hybridization reaction is a two-phase reaction, and washing numerous times to remove unhybridized probe. Developing the autoradiogram typically takes 24 hours.
Hybridized probe may also be separated from unhybridized probe using hydroxylapatite and appropriate ionic strength buffers (Kohne, D. E., 1984, Patent Cooperation Treaty WO 84/02721). This method, which has only been demonstrated with radioactive probes, is much faster. There is no binding of target DNA to an immobilization support, no blocking of binding sites on a support, fewer washes, and hybridization is more rapid since the reaction take place in a single phase. Commercial kits using hydroxylapatite require only 14 steps and 1.5 hours.
The radioactive method has not been well received in medical diagnostics laboratories for several reasons. First, the high specific activity radioactive materials typically used in kits employing this method have a relatively short half life. Consequently, the complementary probe DNA must be prepared just prior to the hybridization procedure. Secondly, the radioactive material creates more rigid handling problems and undesirable hazards. It is therefore advantageous in most cases to provide a label which is less hazardous and prolongs the shelf life of the probe.
The development of non-radioactive labelling of nucleic acid probes presents an alternative. A typical non-radioactive system is based on the incorporation of a biotinylated deoxyuridine triphosphate into the DNA probe by the nick translation procedure. The resultant biotinylated DNA probe is stable and behaves as does a non-biotinylated DNA probe. The biotinylated DNA probe technique has been applied to the detection of specific DNA and RNA sequences in fixed cells or in tissues following in situ hybridizations. It has also been used to visualize probe nucleic acid hybridized to target nucleic acid immobilized on membranes. The detection of the hybridized biotinylated probe is accomplished by either fluorescent antibody or enzyme amplification techniques. A typical non-radioactive labelled probe assay such as an enzyme labelled probe method using membrane-bound target DNA generally requires at least 32 steps and 18 hours. More washes are required than in the case where a radioactive probe is used, but development of the signal is more rapid. Thus, although avoiding the problems of radioactivity, the conventional enzyme-label method is cumbersome in view of the large number of steps and the long test time generally attributable to the above-described immobilization of target nucleic acid on a membrane as the method to effect separation of hybridized from unhybridized probe nucleic acid.
A number of suggestions have been made in the literature for alternative separation methods to recover the hybridized probe target molecules. For example, U.S. Pat. No. 4,599,303, which teaches a method of first hybridizing and then forming covalent bonds between probe and target, discloses at column 2, lines 55-61 thereof, several procedures for separating covalently crosslinked double-stranded probe-target complex from single-stranded probe. These procedures are described as including gel filtration, hydroxylapatite chromatography, enzymatic digestion, alkaline hydrolysis, and photoreversal or chemical reversal of uncrosslinked crosslinking molecules.
The gel filtration technique referred to in the '303 patent generally denotes a column chromatography-type procedure whereby large molecules in the assay mixture pass through channels in the gel, whereas the smaller molecules elute at different velocities through the gel to effect a separation. This gel filtration procedure is believed by the present inventors to be slow, cumbersome, and subject to failure since, with sufficient elution, the entire assay mixture will pass through the column.
In view of the above, new methods for separating single-stranded probe nucleic acid from double-stranded probe target nucleic acid molecules are expected to be highly desired in a commercial setting, particularly in the medical diagnostics and environmental monitoring fields. More specifically, the development of a simpler such technique employable with either radioactive or non-radioactive labelled probes for clinical diagnosis and environmental monitoring would undoubtedly enhance this technique and will have a competitive advantage relative to current approaches.